1. Field of the Invention
This invention relates to certain growth hormone variants, and pegylated forms thereof, for use as agonists or antagonists of human growth hormone.
2. Description of the Related Art
Human growth hormone (hGH) participates in much of the regulation of normal human growth and development. This 22,000-dalton pituitary hormone exhibits a multitude of biological effects, including linear growth (somatogenesis), lactation, activation of macrophages, and insulin-like and diabetogenic effects, among others. Chawla, Annu. Rev. Med., 34: 519 (1983); Edwards et al., Science, 239: 769 (1988); Isaksson et al., Annu. Rev. Physiol., 47: 483 (1985); Thorner and Vance, J. Clin. Invest., 82: 745 (1988); Hughes and Friesen, Annu. Rev. Physiol., 47: 469 (1985). These biological effects derive from the interaction between hGH and specific cellular receptors. Growth hormone deficiency in children leads to dwarfism, which has been successfully treated for more than a decade by exogenous administration of hGH. There is also interest in the antigenicity of hGH to distinguish among genetic and post-translationally modified forms of hGH (Lewis, Ann. Rev. Physiol., 46: 33 [1984]), to characterize any immunological response to hGH when it is administered clinically, and to quantify circulating levels of the hormone.
hGH is a member of a family of homologous hormones that include placental lactogens, prolactins, and other genetic and species variants of growth hormone. Nichol et al., Endocrine Reviews, 7: 169 (1986). hGH is unusual among these in that it exhibits broad species specificity and binds to either the cloned somatogenic (Leung et al., Nature, 330: 537 [1987]) or prolactin (Boutin et al., Cell, 53: 69 [1988]) receptor. The cloned gene for hGH has been expressed in a secreted form in E. coli (Chang et al., Gene, 55: 189 [1987]) and its DNA and amino acid sequences have been reported. Goeddel et al., Nature, 281: 544 (1979); Gray et al., Gene, 39: 247 (1985). The three-dimensional folding pattern for porcine growth hormone (pGH) has been reported at moderate resolution and refinement. Abdel-Meguid et al., Proc. Natl. Acad. Sci. USA, 84: 6434 (1987). The receptor and antibody epitopes of hGH have been identified by homolog-scanning mutagenesis and alanine-scanning mutagenesis as described in the priority application to this application and in Cunningham et al., Science, 243: 1330-1336 (1989) and Cunningham and Wells, Science, 244: 1081-1085 (1989).
There are a large number of high-resolution structures that show the molecular details of protein--protein interfaces (for reviews, see Argos, Protein Eng., 2: 101-113 [1988]; Janin et al., J. Mol. Biol., 204: 155-164 [1988]; Miller, Protein Eng., 3: 77-83 [1989]; Davies et al., Annu. Rev. Biochem., 59: 439-473 [1990]). These define contact residues, but not the energetics for them nor do they show how docking occurs. A comprehensive understanding of the role of contact residues in affecting association and dissociation is fundamental to molecular recognition processes, and is practically important for the rational protein and drug design.
Perhaps the best characterized hormone-receptor complex is that between hGH and the extracellular domain of its receptor (hGHbp). For a review, see Wells and De Vos, Annu. Rev. Biophys. Biomol. Struct., 22: 329-351 (1993). High-resolution structural and mutational analysis (Cunningham and Wells, supra; Cunningham et al., Science, 254: 821-825 [1991]) and structural analysis (De Vos et al., Science,255: 306-312 [1992]) has shown that one molecule of hGH binds two receptor molecules sequentially using distinct sites on the hormone, called Sites 1 and 2.
A number of naturally occurring mutants of hGH have been identified. These include hGH-V [Seeberg, DNA, 1: 239 (1982); U.S. Pat. Nos. 4,446,235, 4,670,393, and 4,665,180] and 20K hGH containing a deletion of residues 32-46 of hGH. Kostyo et al., Biochem. Biophys. Acta, 925: 314 (1987); Lewis et al. J. Biol. Chem., 253: 2679 (1978).
One investigator has reported the substitution of cysteine at position 165 in hGH with alanine to disrupt the disulfide bond which normally exists between Cys-53 and Cys-165. Tokunaga et al., Eur. J. Biochem., 153: 445 (1985). This single substitution produced a mutant that apparently retained the tertiary structure of hGH and was recognized by receptors for hGH.
Another investigator has reported the in vitro synthesis of hGH on a solid resin support. The first report by this investigator disclosed an incorrect 188 amino acid sequence for hGH. Li et al., J. Am. Chem. Soc., 88: 2050 (1966); U.S. Pat. No. 3,853,832. A second report disclosed a 190-amino acid sequence. U.S. Pat. No. 3,853,833. This latter sequence is also incorrect. In particular, hGH has an additional glutamine after position 68, a glutamic acid rather than glutamine at position 73, an aspartic acid rather than asparagine at position 106, and an asparagine rather than aspartic acid at position 108.
In addition to the foregoing, hybrid interferons have been reported that have altered binding to a particular monoclonal antibody. Camble et al., "Properties of Interferon-.alpha.2 Analogues Produced from Synthetic Genes" in Peptides: Structure and Function. Proceedings of the Ninth American Peptide Symposium, Deber et al., eds. (Pierce Chemical Co., Chicago, Ill., 1985), pp. 375-384. As disclosed therein, amino acid residues 101-114 from .alpha.-1 interferon or residues 98-114 from .gamma.-interferon were substituted into .alpha.-2 interferon. .alpha.-2 interferon binds NK-2 monoclonal antibody, whereas .alpha.-1 interferon does not. This particular region in .alpha.-2 interferon apparently was chosen because 7 of the 27 amino acid differences between .alpha.-1 and .alpha.-2 interferon were located in this region. The hybrids so obtained reportedly had substantially reduced activity with NK-2 monoclonal antibody. When tested for antiviral activity, such hybrids demonstrated antiviral activity on a par with the activity of wild-type .alpha.-2 interferon. Substitutions of smaller sections within these regions were also reported. Sequential substitution of clusters of 3 to 7 alanine residues was also proposed. However, only one analog [Ala-30,32,33] IFN-.alpha.2 was disclosed.
Alanine substitution within a small peptide fragment of hen egg-while lysozyme and the effect of such substitutions on the stimulation of 2A11 or 3A9 cells has also been reported. Allen et al., Nature, 327: 713-715 (1987).
Others have reported that binding properties can be engineered by replacement of entire units of secondary structure including antigen binding loops (Jones et al., Nature, 321: 522-525 [1986]) or DNA recognition helices. Wharton et al., Nature, 316: 601-605 (1985).
The structure of amino-terminal methionyl bovine growth hormone (bGH) containing a spliced-in sequence of hGH including histidine 18 and histidine 21 has been shown. U.S. Pat. No. 4,880,910. Additional hGH variants are described in the priority applications for this application and in copending U.S. Ser. Nos. 07/715,300 filed Jun. 14, 1991 and 07/743,614 filed Aug. 9, 1991, and WO 92/09690 published Jun. 11, 1992. hGH variants are also disclosed (WO 93/00109 published Jan. 7, 1993) having the GH moiety covalently attached to poly(ethylene glycol) (PEG) at one or more amino acids, including those wherein the PEG molecule is attached to the lysine at position 41.
hGH variants are also reported in WO 92/21029 published Nov. 26, 1992, which discloses the 1:2 complex dimer between GH and two receptor molecules. The variant is a monomeric polypeptide ligand which comprises in its native conformation four amphipathic alpha helices and which binds to its receptor through two sites in sequential order. This variant comprises a mutation introduced into site 1 or site 2, provided that when the ligand is GH, at least two residues are mutated, one each in the N-terminal about 15 residues of the wild-type hormone and in helix C, or site 1 is mutated so as to increase the affinity of the ligand for its receptor at site 1.
It has previously been shown that monovalent phage display (Bass et al., Proteins, 8: 309-314 [1990]) can be used to improve the affinity of Site 1 in hGH for the hGHbp. Lowman et al., Biochemistry, 30: 10832-10838 (1991). Modest improvements in binding affinity (3 to 8-fold tighter than wild-type hGH) were produced by sorting three independent libraries each mutated at four different codons in Site 1. An hGH mutant slightly enhanced in binding affinity for Site 1 and blocked in its ability to bind Site 2 was a better antagonist of the hGH receptor than the Site 2 mutant alone. Fuh et al., Science, 256: 1677-1680 (1992). It would be desirable to improve Site 1 affinity further to obtain an even better antagonist that can have utility in treating conditions of GH excess such as acromegaly.
Additional improvements in Site 1 affinity might be obtained by mutating more residues per library. However, it was not feasible to generate enough transformants to ensure that all possible residue combinations were represented when more than about five codons were randomized simultaneously. Lowman and Wells, Methods: Companion Methods Enzymol., 3: 205-216 (1991). Mutations at protein--protein interfaces usually exhibit additive effects upon binding. Wells, Biochemistry, 29: 8509-8517 (1990).
It is desired to obtain much larger improvements in affinity. It has been disclosed that the lysine residues of hGH and bGH are involved in the interaction of hGH and bGH with somatotropic receptors, with the structure-function relationship particularly implicating the lysine or arginine residues at positions 41, 64, 70, and 115. Martal et al., FEBS Lett., 180: 295-299 (1985). Lysine residues were chemically modified by methylation, ethylation, guanidination, and acetimidination, resulting in reduced activity by radioreceptor assay.
The in vivo efficacy of hGH and hGH variants is determined, in part, by affinity for hGH receptor and by the rate of clearance from the circulation. The in vivo half-life of certain other therapeutic proteins has been increased by conjugating the proteins with PEG, which is termed "pegylation." See, e.g., Abuchowski et al., J. Biol. Chem., 252:3582-3586 (1977). PEG is typically characterized as a non-immunogenic uncharged polymer with three water molecules per ethylene oxide monomer. PEG is believed to slow renal clearance by providing increased hydrodynamic volume in pegylated proteins. Maxfield et al., Polymer, 16:505-509 (1975). In one study, Katre and co-workers (Knauf, M. J. et al., J. Biol. Chem., 363:15064-15070 [1988]; Goodson, R. J. & Katre, N. V., Bio/Technology, 8:343-346 [1990]) showed that the in vivo half-life of PEG-interleukin-2 increased with effective molecular weight. In addition, pegylation has been reported to reduce immunogenicity and toxicity of certain therapeutic proteins. Abuchowski et al., J. Biol. Chem., 252:3578-3581 (1977).